9014
Biochemistry 2001, 40, 9014-9022
DNA Polymerase β: Pre-Steady-State Kinetic Analyses of dATPRS Stereoselectivity and Alteration of the Stereoselectivity by Various Metal Ions and by Site-Directed Mutagenesis† Jia Liu‡ and Ming-Daw Tsai* Departments of Biochemistry and Chemistry, Ohio State Biochemistry Program, and Protein Research Group, The Ohio State UniVersity, Columbus, Ohio 43210 ReceiVed March 30, 2001; ReVised Manuscript ReceiVed June 5, 2001
ABSTRACT: The first pre-steady-state kinetic analysis of the stereoselectivity of a DNA polymerase, Pol β from rat brain, toward Rp and Sp isomers of dATPRS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPRS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (kpol) and the apparent dissociation constant (Kd,app) were measured with dATP, Rp-dATPRS, and Sp-dATPRS in the presence of Mg2+, Mn2+, or Cd2+. The results indicate that wild type (WT) polymerase (Pol) β can incorporate both Sp- and RpdATPRS in the presence of Mg2+, but Sp is the preferred isomer. The stereoselectivity, defined as (kpol/ Kd)Sp/(kpol/Kd)Rp (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg2+. When Mg2+ was substituted with Mn2+ and Cd2+, the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol β complexes, as well as previous steady-state kinetic studies of other DNA polymerases. In addition, the D276R mutant was designed to introduce a potential extra hydrogen bonding interaction between the arginine side chain and the pro-Sp oxygen of the R-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPRS (Sp/Rp ratio ) 1.5, 3.7, and 1.5 for Mg2+, Mn2+, and Cd2+, respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPRS relative to WT Pol β, suggesting that this mutant Pol β can be used to incorporate Rp-dNTPRS into DNA oligomers.
DNA polymerase β (Pol β)1 catalyzes the template-directed nucleotidyl transfer reaction and performs an essential part of the DNA repair process in filling single-stranded gaps in the base excision repair process (2-4). Extensive kinetic studies and in vivo mutation assays of the wild-type protein and different mutants have been performed to elucidate the catalytic mechanism and fidelity of Pol β (5-10). Several crystal structures, including binary and ternary complexes of Pol β, have been solved in the past several years (1113). Like most other polymerases, Pol β consists of three distinct subdomains (designated thumb, palm, and fingers), forming a DNA binding channel. It should be noted that in our earlier publications we have followed the designation of thumb and fingers subdomains of Pelletier et al. (11), whereas in the present paper we switch to that of Steitz et † This work was supported by NIH Grant GM43268. This is paper 9 in the series “DNA Polymerase β”. For paper 8, see Arndt et al. (1). * Author to whom correspondence should be addressed at the Department of Chemistry [telephone: (614) 292-3080; fax (614) 2921532; e-mail
[email protected]]. ‡ Department of Biochemistry. 1 Abbreviations: BSA, bovine serum albumin; dAMP, 2′-deoxyadenosine 5′-O-phosphate; dAMPS, 2′-deoxyadenosine 5′-O-phosphorothioate; dATPRS, 2′-deoxyadenosine 5′-O-(1-thiotriphosphate); dNTP, 2′-deoxynucleoside 5′-triphosphate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; Hepes, N-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid]; Pol I, Escherichia coli DNA polymerase I; Pol β, rat DNA polymerase β; RT, reverse transcriptase; S-oligos, phosphorothioate analogues of oligonucleotides; TLC, thin-layer chromatography; Tris, tris(hydroxymethyl)aminomethane; WT, wild type.
al. (14) to facilitate a more accurate comparison between Pol β and other polymerases. The requirement of divalent metal ions for catalysis was first recognized in the early study of Escherichia coli DNA polymerase I (Pol I) (15). Subsequent studies with other polymerases confirmed that Mg2+ is likely the divalent metal ion utilized by most polymerases for catalysis in vivo (16). On the basis of the structures of ternary complexes of Pol β with DNA and ddCTP substrates (13), specific roles have been assigned for two Mg2+ ions in the active site of Pol β as shown in Figure 1. Recently we have shown that binding of Cr(III)dNTP alone (before binding of the second metal ion) to Pol β is sufficient to induce the closing of the fingers subdomain (1). Stereoselectivity of an enzyme toward Rp and Sp isomers of phosphorothioate analogues of dNTP, coupled with variation of metal ions, has long been established as a useful tool in probing detailed interactions between metal ions and nucleotides at the active site of enzymes (17-21). However, this approach has not been used for DNA polymerases, except for early steady-state kinetic studies of E. coli Pol I (22) and phage T4 DNA polymerase (23). The results of these studies indicated that DNA polymerases accept the Sp isomer of dNTPRS exclusively, even when Mg2+ was replaced by Co2+ or Mn2+, which has a higher preference for sulfur ligands. As pointed out by Pelletier et al. (24), this result appeared to be in contradiction with the observa-
10.1021/bi010646j CCC: $20.00 © 2001 American Chemical Society Published on Web 07/04/2001
dATPRS Steroselectivity of DNA Polymerase β
FIGURE 1: Active site of Pol β showing the interactions of the incoming nucleotide with the three aspartic acid residues and the two metal ions (13).
tion that when dATPRS was cocrystallized with Pol β in the presence of Mn2+, the Rp isomer instead of the Sp isomer binds to the active site. In this paper we report detailed analyses of the activities of Pol β toward Rp and Sp isomers of dATPRS using pre-steady-state kinetics, seeking to elucidate the stereoselectivity and to understand the metal ion binding properties of Pol β. The second goal of this paper is to construct mutants of Pol β that will show improved activity toward the disfavored Rp isomer of dATPRS. If a Pol β mutant with improved Rp isomer activity can be obtained, it could potentially lead to the synthesis of “phosphorothioate analogues of oligonucleotides” (abbreviated “S-oligos”) from the Rp isomer of dNTPRS. The potential significance of this goal is addressed under Discussion. As an effort toward this goal, the D276R mutant was designed to introduce an extra interaction between the arginine side chain of the enzyme and the R-phosphate of dNTP. Such an interaction, if actually occurring, could perturb the preference of the enzyme toward isomers of dNTPRS. The results indicate that the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPRS and a 5-fold enhanced reactivity toward the Rp isomer relative to WT Pol β. MATERIALS AND METHODS Materials. Ultrapure dNTP, dAMPS, dATPRS mixed isomers, nuclease, and protease activity free BSA and G-25 microspin columns were obtained from Pharmacia Biotech. Thermostable Pfu DNA polymerase was from Stratagene. [γ-32P]ATP was from ICN Biomedicals. T4 polynucleotide kinase, pyruvate kinase, adenylate kinase, and DpnI exonuclease were purchased from New England Biolabs. Mutagenesis and Enzyme Preparation. By use of appropriate mutagenic oligonucleotides (IDT Inc.) and wildtype Pol β plasmid [pET-17b(Pol β)] (25) as a template, Pol β mutant D276R was created by the “QuikChange” method according to the protocol from Stratagene. The resulting mutation was confirmed by sequencing the entire Pol β gene according to the Sanger method using a 373A DNA sequencer (Applied Biosystems). WT Pol β and mutant Pol
Biochemistry, Vol. 40, No. 30, 2001 9015 β were then overexpressed in an E. coli system, BL21(DE3)[plysS, pET-17b(Pol β)] (25) and purified as described previously (8). The enzymes were estimated to be >90% homogeneous on the basis of SDS-PAGE analysis developed by the silver-staining method. The exact concentrations of the enzyme solutions were determined by using an extinction coefficient of 21200 M-1 cm-1 at 280 nm (26). Preparation of Pure Stereoisomers. Sp-dATPRS was synthesized from dAMPS by coupled reactions of adenylate kinase and pyruvate kinase. The reaction mixture contained in 40 mL at room temperature 7.5 mM ATP, 5.0 mM dAMPS, 15 mM phosphoenol pyruvate, 50 mM KCl, 15 mM MgCl2, 30 mM Tris-HCl buffer (pH 8.0), 1 mM DTT, 200 units/mL adenylate kinase, and 10 units/mL pyruvate kinase. The pH of the reaction mixture was adjusted to 8.0 with 0.3 M potassium hydroxide before the enzymes were added. The reaction was followed by thin-layer chromatography on silica gel plates containing a fluorescent indicator with a 1.0 M lithium chloride buffer system. Nucleotides were visualized with UV light. The reaction mixture was then applied to a DEAE-Sephadex A-25 column at 4 °C with a linear gradient of 1.0 L of 0.2 M and 1.0 L of 0.75 M triethylammonium bicarbonate buffers (pH 7.5). The fractions that contained dATPRS were pooled and lyophilized to dryness. Finally, the proton-decoupled 31P NMR was used to check the purity of the product. Rp-dATPRS was obtained from dATPRS mixed isomers by removing the Sp-dATPRS using adenylate kinase (27). The incubation mixture (total volume of 6 mL) contained 3 mM dATPRS (mixture of isomers), 6 mM dAMP, 10 mM DTT, and 50 mM Tris-HCl (pH 8.0). About 300 units of adenylate kinase (100 µL, dialyzed against 10 mM Tris-HCl, pH 8.0, for 2 h prior to use) was added to the reaction mixture. The reaction was followed by TLC as described for the Sp isomer preparation. After 2 h of incubation at room temperature, ∼50% dATPRS mixed isomers had been consumed and the reaction mixture was chromatographed on a DEAE-Sephadex column with a linear gradient of 500 mL each of 0.1 and 0.6 M triethylammonium bicarbonate. DNA Substrates. DNA oligomers were purchased from IDT Inc. (Coralville, IA). Template/primer DNA substrates (annealed heteropolymeric complementary [45mer template]/ [25mer primer] duplexes) were used in the experiments. Both oligonucleotides were purified by electrophoresis through denaturing (7 M urea) polyacrylamide gels; a 16% gel was used to purify the 25mer and a 10% gel for the 45mer. Sequences of the substrates used can be found in Werneburg et al. (25), and the substrates were prepared as described (25). The 25mer primer was 5′-radiolabeled with [γ-32P]ATP (4500 Ci/mmol) and T4 polynucleotide kinase as described in the manufacturer’s protocol. The unreacted [γ-32P]ATP was removed from the 5′-radiolabeled DNA solution with a G-25 microspin column (Pharmacia). The labeled primer DNA was mixed with ∼100-fold molar excess of unlabeled DNA (primer and template DNA), and the T4 polynucleotide kinase was inactivated at 80 °C for 5 min. The solution was slowly cooled to room temperature for 2 h to anneal the DNA substrate. Kinetic Experiments and Product Analysis. A rapid quench instrument (KinTek Instrument Corp., State College, PA) was used for rapid quench experiments with reaction times
9016 Biochemistry, Vol. 40, No. 30, 2001
Liu and Tsai
FIGURE 2: Preparation of pure Sp- and Rp-dATPRS diastereomers by enzymatic methods: (A) preparation of Sp isomer from AMPS by the coupled reactions of adenylate kinase (AK) and pyruvate kinase (PK) (PEP, phosphoenol pyruvate); (B) preparation of the Rp isomer by AK-catalyzed consumption of the Sp isomer from the mixture.
ranging from 10 ms to 20 s. The typical experiment was performed at 37 °C in 50 mM Tris-HCl (pH 8.0) containing 50 mM KCl, 10% glycerol, 5.0 mM MgCl2, and various [dNTP]. All concentrations given refer to the final concentrations after mixing. Pol β was preincubated with the DNA substrate prior to rapid mixing with dNTP and divalent metal ions to begin the reaction that was quenched with 0.5 M EDTA. For reaction times >20 s, 20 µL aliquots of the reaction mixture (normally total volume of 300 µL) were removed manually and the reactions were stopped by the addition of 0.5 M EDTA (pH 8.0) after the selected time intervals. The quenched samples were mixed with an equal volume of gel loading buffer and denatured at 85 °C for 5 min, and the products were separated on a 16% polyacrylamide-7 M urea gel. The disappearance of substrate (25mer) and the formation of product (26mer) were analyzed with a STORM 840 PhosphorImager (Molecular Dynamics) to quantify product formation. A ∼10-fold excess of the enzyme (1 µM in the reaction mixture) relative to the DNA substrate (100 nM) was used for all assays. Under these conditions, >90% of the DNA substrate should be complexed to the enzyme (25), and the reaction time course is represented by a characteristic burst phase of dNTP incorporation only. Thus, the rate of single catalytic turnover is measured. Data Analysis. Data obtained from kinetic assays were fitted by nonlinear regression using Sigma Plot software (Jandel Scientific) with the appropriate equations. The apparent burst rate constant (kobs) for each particular concentration of dNTP was determined by fitting the time courses for the formation of product (26mer) with eq 1
[26mer] ) A[1 - exp(-kobst)]
(1)
where A represents the burst amplitude. The turnover number kpol and apparent dissociation constant for dNTP (Kd,app) were then obtained from plotting the apparent catalytic rates, kobs, against dNTP concentrations and fitting the data with the hyperbolic eq 2
kobs ) (kpol[dNTP])/([dNTP] + Kd,app)
(2)
RESULTS Preparation of Diastereomeric Phosphorothioate Analogues. We used enzymatic methods to synthesize pure
FIGURE 3: 31P NMR spectrum for dATPRS (10 mM Tris, 1 mM EDTA buffer, pH 7.5): (A) purified Rp isomer; (B) purified Sp isomer; (C) mixed isomers. Only the R-phosphate region of the spectrum is shown.
dATPRS isomers to very high purity, as illustrated in Figure 2. Sp-dATPRS was synthesized from dAMPS by the coupled reactions of adenylate kinase and pyruvate kinase in a procedure modified from that of Brody and Frey (28). RpdATPRS was purified from commercially available mixed isomers by removing the Sp-dATPRS using adenylate kinase (27). The proton-decoupled 31P NMR spectra of purified Sp and Rp isomers are shown in Figure 3 (only PR regions are shown). No detectable peak from the other isomer was present in either spectrum, and the purity was estimated to be >98% for both isomers on the basis of the signal-tonoise ratio. Pre-Steady-State Kinetic Analyses of WT Pol β in the Presence of Different Metal Ions. Pre-steady-state kinetic experiments were first performed to determine the ability of different metal ions to replace Mg2+, as well as the optimal concentration of each metal ion. As shown in Figure 4, Mn2+
dATPRS Steroselectivity of DNA Polymerase β
FIGURE 4: Dependence of Pol β activities on Mg2+ (A), Mn2+ (B), or Cd2+ (C). The WT Pol β preincubated with DNA substrate was mixed with metal ion and dATP (200 µM) solutions, and the reaction was quenched after an allotted time. The results were fitted to a single-exponential equation (eq 1) to obtain kobs for each metal ion concentration. The observed catalytic rate (kobs) was then plotted against metal ion concentration. Tris buffer (pH 8.0, 50 mM) was used for Mg2+ and Mn2+, whereas Hepes buffer (pH 8.0, 50 mM) was used for Cd2+.
can activate Pol β as well as Mg2+ does at low concentrations (2 mM, whereas Mg2+ reaches its optimal concentration at 9 mM. Pol β activity with Cd2+ (0.1-1 mM) in the typical Tris buffer used for other metal ions was extremely low, and almost no turnover was detected during the reaction time (up to 30 min). However, in 50 mM Hepes buffer (pH 8.0) complete turnover of the DNA substrates was observed under single-turnover condition, although with a much lower activity (∼100 fold slower than that with Mg2+) and very low optimal concentration (0.1 mM). We then chose the metal ion concentrations that gave optimal activity (1 mM Mn2+ and 0.1 mM Cd2+) or close to optimal activity (5 mM Mg2+, 85% optimal activity) for the following studies with different metal ions as well as dNTPRS isomers of WT and mutant Pol β. The rate of polymerization (kpol) and the apparent dissociation constant of dATP (Kd,app) were determined by
Biochemistry, Vol. 40, No. 30, 2001 9017
FIGURE 5: Pre-steady-state kinetics of Sp-dATPRS incorporation catalyzed by WT Pol β with Mg2+ as the divalent metal ion. The reaction conditions and methods of product analysis were as described under Materials and Methods. (A) Product-time plot. DNA substrate (100 nM) was incubated with 1000 nM Pol β and reacted with 1, 2, 5, 12.5, 25, and 50 µM Sp-dATPRS diastereomeric isomer. The reaction was quenched with 0.5 M EDTA after 0.02, 0.04, 0.08, 0.16, 0.3, 0.6, 1, 2, and 4 s. The results were fitted to a single-exponential equation (eq 1) to obtain kobs for each SpdATPRS concentration. (B) Sp-dATPRS concentration dependence curve. kpol and Kd,app values were determined as 5.3 ( 0.2 s-1 and 3.3 ( 0.4 µM, respectively, by fitting to the hyperbolic equation (eq 2).
measuring the concentration dependence of dATP incorporation on the rate of the 25/45mer DNA substrate by Pol β under single-turnover conditions. Representative data for the determination of kpol and Kd,app are shown in Figure 5. The results of the pre-steady-state kinetic parameters of WT Pol β with dATP in the presence of different metal ions are summarized in Table 1. In the presence of Mn2+ Pol β shows kpol and Kd,app values very similar to those in the presence of Mg2+, whereas Cd2+ shows a much lower activity (400-fold decrease in kpol) but a tighter binding of dATP (20-fold lower Kd,app). StereoselectiVity of WT Pol β toward Stereoisomers of dATPRS in the Presence of Different Metal Ions. We next compared kpol and Kd,app of WT Pol β toward dATPRS diastereomers in the presence of different metal ions. The ratio kpol/Kd,app denotes the substrate specificity of the enzyme. The data are summarized in Table 2 and indicate that the kpol values for dATPRS isomers are lower than the corresponding values for dATP by factors of 2-50. However, the Kd,app values are also significantly lower for dATPRS,
9018 Biochemistry, Vol. 40, No. 30, 2001
Liu and Tsai
Table 1: Pre-Steady-State Kinetic Parameters of WT Pol β and Mutant D27R with dATP Substrates in the Presence of Different Metal Ions enzyme
metal ion
kpol (s-1)
Kd,app (µM)
kpol/Kd,app (M-1 s-1)
WTa WTa WTb D276R D276R D276R
Mg2+ Mn2+ Cd2+ Mg2+ Mn2+ Cd2+
26.4 ( 0.9 19.2 ( 0.7 0.065 ( 0.0046 8.6 ( 0.87 2.3 ( 0.10 NDc
32.2 ( 4.1 27.9 ( 2.6 2.6 ( 0.90 13 ( 4.3 3.6 ( 0.58 ND
820000 688000 25000 660000 640000 ND
a
These data differ slightly from those published previously (5, 25) due to the different conditions used in this study. b All reactions with Cd2+ as divalent metal cation were performed in 50 mM Hepes buffer at pH 8.0. All other reactions with Mg2+ or Mn2+ were performed in 50 mM Tris buffer at pH 8.0. c There was no detectable turnover for D276R mutant with Cd2+ as the divalent cation. Table 2: Pre-Steady-State Kinetic Parameters of WT Pol β and Mutant D276R with dATPRS Diastereomers in the Presence of Different Metal Ions enzyme
metal stereoion isomer
WT WT WT WT WT WT D276R D276R D276R D276R D276R D276R
Mg2+ Mg2+ Mn2+ Mn2+ Cd2+ Cd2+ Mg2+ Mg2+ Mn2+ Mn2+ Cd2+ Cd2+
Sp Rp Sp Rp Sp Rp Sp Rp Sp Rp Sp Rp
kpol (s-1)
Kd,app (µM)
kpol/Kd,app (M-1 s-1)
5.32 ( 0.20 3.3 ( 0.4 1610000 0.63 ( 0.033 22.3 ( 3.1 28000 0.69 ( 0.025 1.1 ( 0.23 630000 0.38 ( 0.038 4.6 ( 1.3 83000 0.047 ( 0.0012 1.2 ( 0.13 39000 0.010 ( 0.002 5.6 ( 1.8 1800 0.95 ( 0.09 5.0 ( 0.79 190000 0.3 ( 0.033 2.4 ( 0.19 130000 0.16 ( 0.0027 0.71 ( 0.075 220000 0.13 ( 0.005 2.2 ( 0.41 59000 0.0034 ( 0.00053 23 ( 9.6 150 0.0011 ( 0.00072 10.6 ( 0.25 100
Table 3: Substrate Stereoselectivity of WT Pol β and Mutant D276R toward dATPRS Diastereomers enzyme
metal ion
(Sp/Rp)kpol
(Sp/Rp)Kd,app
(kpol/Kd,app)Sp/ (kpol/Kd,app)Rp
WT WT WT D276R D276R D276R
Mg2+ Mn2+ Cd2+ Mg2+ Mn2+ Cd2+
8.4 1.8 4.7 3.2 1.2 3.1
0.15 0.24 0.21 2.1 0.32 2.2
57.5 7.6 21 1.5 3.7 1.5
with the exception of the Rp isomer in the presence of Cd2+. As a result, Sp-dATPRS is a slightly better substrate (higher kpol/Kd,app ratio) than dATP for WT Pol β in the presence of Mg2+. The stereoselectivity, defined as (kpol/Kd,app)Sp/(kpol/Kd,app)Rp (abbreviated the Sp/Rp ratio), is shown in Table 3. Overall, WT Pol β accepts both isomers but with a preference toward the Sp isomer. The Rp isomer displays lower kpol and higher Kd values for all three metal ions. The Sp/Rp ratios are 57.5, 7.6, and 21, respectively, in the presence of Mg2+, Mn2+, and Cd2+. These data indicate that, although substitution of Mg2+ by the “softer” metal ions (which prefer sulfur ligands over oxygen ligands in the order Mg2+ < Mn2+ < Cd2+) (29) caused some relaxation of stereoselectivity, it did not follow the expected order and did not lead to a reversal of stereoselectivity. Pre-Steady-State Kinetic Analyses of D276R Mutant in the Presence of Different Metal Ions. The stereoselectivity of Pol β toward dNTPRS isomers should be dictated mostly
by the interactions between the R-phosphate of dNTP and enzymatic residues or metal ions. The crystal structures of ternary complexes of Pol β show no direct interaction between the enzyme and the nonbridging oxygens of the R-phosphate of dNTP (11, 13). On the basis of the crystal structure and protein modeling, we predict that replacing Asp276 with Arg will likely introduce a new hydrogen bonding between the enzyme and the pro-Sp oxygen of the R-phosphate of dNTP and that such a new interaction will likely perturb the stereoselectivity of Pol β. The D276R mutant was then constructed to test whether the stereoselectivity of Pol β toward dATPRS isomers can be changed. First, we measured the catalytic activity and dissociation constant of regular nucleotide dATP for this mutant protein in the presence of different metal ions. As shown in Table 1, D276R displays a small decrease (by a factor of 3) in kpol relative to WT Pol β with Mg2+ as the divalent ion and a moderate decrease (by a factor of 8) with Mn2+. There was no detectable activity for D276R mutant with Cd2+ as the divalent metal ion in the reaction. The Kd,app of dATP of this mutant is also lower than that of WT Pol β, suggesting a tighter binding of nucleotide for the D276R mutant. This supports enhanced interaction between the enzyme and the nucleotide. As a consequence of decreases in both kpol and Kd,app, the substrate specificity kpol/Kd,app toward dATP is comparable between D276R and WT in the presence of Mg2+ or Mn2+. StereoselectiVity of D276R Pol β toward Stereoisomers of dATPRS in the Presence of Different Metal Ions. We then examined the stereoselectivity of the D276R mutant toward dATPRS diastereomers with different metal ions, and the results are summarized in the lower half of Table 2 (kpol and Kd,app values) and Table 3 (Sp/Rp ratios). As shown by the data, the Sp/Rp ratio decreases from 57.5 for WT to 1.5 for D276R in the presence of Mg2+. This result confirms our prediction that the newly introduced positive side chain may interact with the pro-Sp oxygen of R-phosphate and relax the stereoselectivity of Pol β. Most importantly, whereas SpdATPRS is a worse substrate for D276R (kpol/Kd,app ) 1.9 × 105 M-1 s-1) than for WT (kpol/Kd,app ) 1.61 × 106 M-1 s-1), the Rp isomer is a substantially better substrate for D276R (kpol/Kd,app ) 1.3 × 105 M-1 s-1) than for WT (kpol/Kd,app ) 2.8 × 104 M-1 s-1). The enhancement in the reactivity of the Rp isomer is mainly due to enhanced bindingsa decrease in Kd by a factor of 10. As shown in Table 3, the stereoselectivities of the D276R mutant in the presence of Mn2+ and Cd2+ are also close to 1. DISCUSSION This work represents the first use of pre-steady-state kinetics to study the metal ion dependence and dNTPRS stereoselectivity for a DNA polymerase, as well as the first example of altering the Sp- vs Rp-dNTPRS stereoselectivity via site-directed mutagenesis. The potential significance of this work is elaborated below. Comparison of dATPRS StereoselectiVity between Pol β and Pol I. Although the results of pre-steady-state kinetics cannot be compared directly with those of steady-state kinetic analyses, it is useful to compare the stereoselectivity of Pol β from this work with that of Pol I in a previous work (22). In both studies, the enzyme was shown to prefer the Sp
dATPRS Steroselectivity of DNA Polymerase β isomer of dATPRS in the presence of Mg2+. However, Pol I showed
